Oil biosynthesis in a cell generically refers to the synthesis of triacylglycerols (TAGs), wherein TAGs are defined as neutral lipids consisting of three fatty acyl residues esterified to a glycerol molecule. Such oils can contain a wide spectrum of fatty acids, including saturated and unsaturated fatty acids and short-chain and long-chain fatty acids. And, not surprisingly, numerous factors affect the quantity of oil so produced and its final fatty acid composition within a specific microbe.
Although traditional approaches (e.g., breeding) and genetic engineering approaches have been successfully applied to produce oilseed plants that have improved oil content [demonstrated by the commercial availability of e.g., high-laurate canola, high-stearate canola, high-oleic soybean and high-oleic corn], similar manipulation of oil content in oleaginous microbes has not been significantly pursued in the past. Recent efforts to engineer microbes having the ability to commercially produce long-chain ω-3 and/or ω-6 polyunsaturated fatty acids (“PUFAs”; e.g., 18:3, 18:4, 20:3, 20:4, 20:5, 22:6 fatty acids) within their oil fraction, however, has created a need for methods to increase carbon flow into lipid metabolism.
Lipid metabolism in most organisms is catalyzed by a multi-enzyme fatty acid synthase complex (“FAS”) and initially occurs by the condensation of eight two-carbon fragments (acetyl groups from acetyl-CoA) to form palmitate, a 16-carbon saturated fatty acid (Smith, S. FASEB J., 8(15):1248-59 (1994)). Once free palmitate (16:0) is released from FAS, the molecule undergoes either elongation (i.e., via a C16/18 fatty acid elongase to produce stearic acid (18:0)) or unsaturation (i.e., via a Δ9 desaturase to produce palmitoleic acid (16:1)). All other fatty acid molecules are synthesized from these two metabolic precursors. Since the primary fate of palmitate is elongation, however (while desaturation is only a minor reaction in most organisms), it is concluded that C16/18 fatty acid elongases play an important role in determining overall carbon flux into the fatty acid biosynthetic pathway, and thereby play a determinant role in both the quantity and composition of oil so produced.
A variety of fatty acid elongases have been isolated and characterized in recent years. For example, a useful review discussing the elongation of long-chain fatty acids in yeast, mammals, plants and lower eukaryotes is that of Leonard, A. E., et al. (Prog. Lipid Res. 43:36-54 (2004)). Table 1 of Leonard et al. provides a summary of fatty acid elongase genes, GenBank Accession Nos. and the reaction that each enzyme catalyzes (i.e., from S. cerevisiae [ELO1, ELO2, ELO3], human, mouse [Elov1, Elov2, Elov3, Elov4, Lce], rat [rELO1, rELO2], Caenorhabditis elegans [CEELO1], Mortierella alpina [GEELO, MAELO], Isochrysis galbana [IgASE1] and Physcomitrella patens [PSE1]). Additional fatty acid elongases that have been described and functionally characterized include those from: Ostreococcus tauri [OtELO1, OtELO2], Thalassiosira pseudomana [TpELO1, TpELO2], Xenopus laevis [XIELO] and Oncorhynchus mykiss [OmELO] (Meyer, A., et al., J. Lipid Res. 45(10):1899-1909 (2004)); Thraustochytrium aureum (U.S. Pat. No. 6,677,145); P. patens [pavELO] (Pereira, S. et al., Biochem. J. 384:357-366 (2004)); and Caenorhabditis elegans CeELO2 (Kniazeva, M. et al., Genetics 163:159-169 (2003)). However, only the rat rELO2 and C. elegans CeELO2 elongases are classified as C16/18 fatty acid elongases having the appropriate substrate specificity to enable conversion of palmitate to stearic acid. Thus, it was desirable herein to identify and characterize a novel C16/18 fatty acid elongase as a means to permit the up-regulation of carbon flow into lipid metabolism in an oleaginous microbe using the techniques of genetic engineering.
Applicants have solved the stated problem by isolating the gene encoding a C16/18 fatty acid elongase from Mortierella alpina and demonstrating increased conversion of 16:0 to 18:0 upon over-expression of the gene in the oleaginous yeast, Yarrowia lipolytica. This enabled increased PUFA content in the microbial oil and increased oil biosynthesis.